Tremella fuciformis is a dimorphic fungus that can undertake a reversible transition between yeast-like conidia and hyphal forms. The transformation mechanism and proteomic differences between these two forms have not been reported. Therefore, in this study, we attempted to explore the differential protein profiles of dikaryotic yeast-like conidia from fruiting bodies and mycelia (FBMds) and dikaryotic mycelia (DM) by synthetically applying high-resolution MS1-based quantitative data-independent acquisition (HRMS1-DIA) full proteomics and parallel reaction monitoring (PRM) targeted proteomics. The results showed that a total of 5687 proteins were quantified, and 2220 of them (39.01%) showed more than a two-fold change in expression. The functional analysis of the differentially expressed proteins (DEPs) confirmed that the DEPs were mainly located in the membrane and nucleus. The FBMds tended to express proteins involved in biosynthesis, metabolism, DNA replication and transcription, and DNA damage repair. At the same time, DM exhibited an increased expression of proteins involved in signal transduction mechanisms such as the mitogen-activated protein kinase (MAPK) signaling pathway and the Ras signaling pathway. Further, phosphorylation analysis confirmed the importance of the MAPK signaling pathway in T. fuciformis dimorphism, and comparative metabolism analysis demonstrated the metabolic difference between FBMds and DM. The information obtained in the present study will provide new insights into the difference between FBMds and DM and lay a foundation for further research on the dimorphism formation mechanism of T. fuciformis.